Detailed accomplishments of each project are summarized below. Sensitization to cockroach allergens is a major risk factor for asthma. The structure of the cockroach allergen Bla g 1 was determined by X-ray crystallography. Using NMR and mass spectrometry (MS), different lipid ligands were found associated with Bla g 1 depending on the source of the protein. Further studies are needed to test whether or not these lipid ligands may contribute to the allergenicity of Bla g 1. Various phospholipids are known to bias the immune response towards a Th0/Th2, contributing to allergic disease. In addition, previous attempts to standardize the units of allergen were impossible due to variable fragmentation patterns of the samples. The structure and subsequent MS work allowed us to understand the unusual fragmentation pattern and standardize the molecular units. This will allow for better measurement of environmental exposure when assessing the risk factors for asthmatic disease. The structure of Bla g 1 was discovered to adopt a previously-uncharacterized protein fold. The structure appears to be a symmetric dimer but in actuality is a repeated genetic sequence that has diverged over time. This suggested to us that the origin of the gene was monomeric, but the genetic information was duplicated to facilitate rapid expression of the full protein. We therefore searched other insect genomes for instances of a monomer and found one such instance in mosquitoes. The monomer contains flanking DNA similar to the related dimers, and RNA-seq data suggests the monomer is expressed in the same situations as the other dimeric relatives. Using our knowledge of the novel structural motif, we were able to model a homodimer structure for the mosquito protein based on the Bla g 1 structure. This appears to be an interesting case in which a combination of genetic, RNA-sequencing, and molecular modeling data has provided insight into the molecular evolution of a protein fold. One of our stated goals has been to map where patient antibodies interact with the allergens in order to suggest modifications for immunotherapy that would generate fewer adverse symptoms in patients. We have been studying a model antibody interacting with Bla g 1, and we have been attempting to crystallize Bla g 1 in complex with this model, which would give direct evidence of the interacting epitope. We have been unable to crystallize the model antibody in complex with the allergen, but we did crystallize the antibody alone. From this we deduced that the binding site on the antibody was highly basic, and hence we generated numerous site-directed mutants of Bla g 1 at primarily acidic sites and tested the effect on binding to the model antibody. From this data we are able to suggest a model for the antibody-interacting epitope of the Bla g 1. Cyclophilin allergens are considered pan-allergens due to their high crossreactivity; i.e. patients sensitized to just one source are usually highly allergic to the allergens from all other sources. This crossreactivity can include autoreactivity where the immune system mistakenly reacts against self-antigens. Indeed, some patients with chronic allergic disease, either asthma or atopic dermatitis, have demonstrated both humoral and cell-mediated autoreactivity. The reason for the crossreactivity is the high sequence identity between members of this protein family. In this period we completed our determination of the structure of the allergen Cat r 1, derived from the rosy periwinkle using NMR techniques. This is the first structure of a cyclophilin protein derived from plants. Using the structure, we have been able to better understand the important residues that likely account for the cross reactivity between plant and mold allergens, and potential residues involved in autoreactivity with human cyclophilins. This knowledge will help in the rational design of immunotherapeutics in that researchers may now design hypo-allergens that also avoid encouraging autoreactivity. Like the cyclophilin allergens, there are GST allergens from many different species but these have received comparatively less attention. A recent paper sparked renewed interest in these allergens due to the demonstrated cross- reactivity of the GST allergen Bla g 5 with a helminth GST from Wucheria bancrofti. A connection between the immune response to helminthes and allergens has long been suspected because humans respond to both with same antibody subtype, IgE. We intend to investigate the cross-reactivity further by determining the structures of Bla g 5, Der p 8, and Blo t 8, all GST-allergens. Further, we intend to compare the cross reactivity of these allergens with a GST from another helminth, Ascaris sp., which we suspect may have similar cross reactivity to the other GST allergens. Using the structures we will be able to compare surface exposed residues and correlate the information with patient cross-reactivity. To date, we have succeeded in determining the structures of Bla g 5, Der p 8, and we have protein for crystallization trials for Blo t 8 and the Ascaris GST. A better understanding of the cross-reactivity will aid clinicians in diagnosis and may suggest various improvements to existing immunotherapies. The protein Ara h 2 is the most potent peanut allergen recognized by >90% of peanut allergic patients. The natural allergen and the recombinant construct used to determine the structure showed different patterns of recognition by patient sera. Based on these comparisons a major site of interaction (an epitope) for about 50% of patients was identified. This success has encouraged us to further map the patient epitopes using a panel of antibodies with various specificities for Ara h 2 and the homologous Ara h 6 allergen. Currently we have selected and produced the Fab fragment of several antibodies and are currently performing crystallization trials with complexes of Ara h 2. Thus far, we have failed to generate quality crystals. We plan to attempt new strategies for crystallization including C-terminal affinity/crystallization tags for Ara h 2. Another problem is the expense of production of the antibodies in hybrodomas for the quantities needed for crystallization. We propose cloning the antibody genes and creating recombinant expression systems in E. coli that will make this production much cheaper. It is our goal to further identify conformational epitopes on peanut allergens in order to better understand the patient response to peanut and to determine whether specific eptiope recognition correlates with any aspect of peanut allergic disease, e.g. risk of anaphylaxis, emergency room visits, or response to oral therapy. To do this we